A research program will be undertaken to study how inteins catalyze and regulate the various steps in protein splicing and protein trans-splicing. Protein splicing is a posttranslational process in which an intervening sequence, termed an intein, is removed from a host protein, the extein. In protein trans-splicing the intein is split into two pieces and splicing only occurs upon reconstitution of these fragments. Inteins are present in unicellular organisms from all 3 phylogenetic domains including several pathogens. In addition, all multicellular organisms contain proteins that undergo autoproteolysis reactions during maturation and that likely catalyze the intramolecular cleavage of peptide bonds in a manner similar to inteins. While we have a reasonable picture of the basic chemical steps in protein splicing, our knowledge of how inteins catalyze and regulate these steps is less well developed. Consequently, there is a need to study the detailed mechanism of the process. This information will not only deepen our understanding of protein splicing and related processes, but will also be critical for the design of splicing inhibitors and for the further development of practical applications of protein splicing. In the first part of the program we propose to test a series of hypotheses (formulated based on work performed in the last funding cycle) related to how inteins coordinate the cascade of chemical steps they catalyze and how trans-splicing inteins interact and fold with high efficiency. Accordingly, we will prepare several intein analogs containing unnatural amino acids, isotopic probes and isopeptide linkages, and then employ these in kinetic, thermodynamic and structural investigations of protein splicing in cis (Aim 1) and in trans (Aim 2). In the Aim 3, we will employ directed protein evolution approaches to isolate new trans-splicing inteins with improved activity and broadened splicing specificities. By acting as protein ligases, these evolved proteins are likely to be of broad utility in protein engineering. However, our primary motivation for generating these tools is to provide a means to generate integral membrane proteins containing defined patterns of isotopic labels, i.e. segmental labeling, for NMR studies. Our initial target will be the K+ channel KcsA (on which we have worked for several years) and segmental labeling will be used to probe aspects of the gating mechanism. Ultimately, we plan to extent this technology to other classes of K+ channel and membrane protein. . PUBLIC HEALTH RELEVANCE Protein splicing is required for the maturation of essential DNA replication and recombination enzymes in several important human pathogens including Mycobacterium tuberculosis (1). In addition, autoprocessing processes closely related to protein splicing are essential for lipid modification of proteins involved in embryonic development and normal tissue homeostasis in all animals, and abnormal activity in these proteins is associated with a variety of disorders in humans (2). The proposed mechanistic investigation of protein splicing will lay the groundwork for the eventual development of inhibitors or modulators of these biomedically relevant processes, as well as the more immediate development of new biotechnology tools.